Severe congenital neutropenia (SCN) is a rare inherited syndrome manifested by impaired granulopoiesis and a marked predisposition to develop acute leukemia. G-CSF treatment, while effective in increasing neutrophil counts in most patients, does not prevent progression to leukemia. Allogeneic bone marrow transplantation, while potentially curative, is associated with significant mortality and morbidity. Thus, there is a clear need for more effective therapies for SCN. There is compelling genetic evidence implicating mutations of the ELA2 gene, encoding neutrophil elastase (NE), as the cause of most cases of SCN. Studies of the molecular mechanisms by which ELA2 mutants induce a block in granulocytic differentiation have been limited by the rarity of SCN and the resulting difficulty in obtaining clinical samples. Instead, most recent studies have focused on ectopic NE expression in immortalized cell lines. However, these cell lines have a limited capacity for differentiation and the level of NE expression may not be physiological. Thus, an accurate animal model of SCN would be an invaluable tool to investigators in this field. We propose to generate transgenic mice in which mutant NE is expressed under the regulatory control of the endogenous ELA2 locus. These mice will be used to explore mechanisms of disease pathogenesis and test novel therapies. A second major objective of this application is to explore the feasibility of RNAi-mediated inhibition of ELA2 gene expression to rescue the block in granulocytic differentiation in primary progenitors from patients with SCN. These studies may ultimately lead to a novel molecularly targeted therapy for SCN. The following specific aims are proposed. 1. We will determine whether expression of mutant NE under the regulatory control of the ELA2 gene locus is sufficient to induce an SCN phenotype in mice. Two complementary state-of-the-art transgenic approaches will be used to generate a mouse model of SCN. 2. We will characterize the effect of RNAi-mediated inhibition of ELA2 gene expression on the growth and differentiation of primary myeloid progenitors. Preliminary data show the feasibility of RNAi to attenuate NE protein expression in cell lines. The efficacy of lentiviral RNAi vectors to inhibit NE protein expression and revert the block in granulocytic differentiation in primary progenitors from patients with SCN will be determined. A novel xenotransplantation model of SCN will be developed to study hematopoiesis in vivo.